Marker assisted selection of bovine for improved milk production using diacylglycerol acyltransferase gene DGAT1

ABSTRACT

The present invention provides a method of genotyping bovine for improved milk production traits by determining the DGAT1 genotypic state of said bovine, wherein the DGAT1 gene and polymorphisms within said gene have been found to be associated with such improved milk production traits.

Reference to Related Applications

The present application is the U.S. national phase of InternationalApplication PCT/NZ/01/00245, filed Oct. 31, 2001, and claims priorityunder 35 U.S.C. §119 to New Zealand Patent Application No. 507888, filedOct. 31, 2000 and New Zealand Patent Application No. 508662 filed Dec.6, 2000.

FIELD OF THE INVENTION

This invention relates to an application of marker assisted selection ofbovine for a quantitative trait loci (QTL) associated with milkproduction, particularly although by no means exclusively, by assayingfor the presence of at least one allele which is associated withincreased milk volume as well as improved milk composition. The presentinvention also relates to the gene associated with the QTL, variouspolymorphisms within the gene sequence, proteins encoded by thesesequences as well as to the application of all of these in the farmingindustry.

BACKGROUND

The genetic basis of bovine milk production is of immense significanceto the dairy industry. An ability to modulate milk volumes and contenthas the potential to alter farming practices and to produce productswhich are tailored to meet a range of requirements. In particular, amethod of genetically evaluating bovine to select those which expressdesirable traits, such as increased milk production and improved milkcomposition, would be desirable.

To date, bovine genomics are poorly understood and little is knownregarding the genes which are critical to milk production. While therehave been reports of quantitative trait loci (QTLs) on bovine chromosome14 postulated to be associated with milk production (Coppieters et al(1998)), the specific genes involved have not to date been identified.

Marker assisted selection, which provides the ability to follow aspecific favourable genetic allele, involves the identification of a DNAmolecular marker or markers that segregate with a gene or group of genesassociated with a QTL. DNA markers have several advantages. They arerelatively easy to measure and are unambiguous, and as DNA markers areco-dominant, heterozygous and homozygous animals can be distinctivelyidentified. Once a marker system is established, selection decisions areable to be made very easily as DNA markers can be assayed at any timeafter a DNA containing sample has been collected from an individualinfant or adult animal, or even earlier as it is possible to testembryos in vitro if such embryos are collected.

The applicants have now identified a gene responsible for the QTL effecton bovine chromosome 14 as well as a number of polymorphisms which areassociated with distinct genetic merits of animals for milk compositionand volume.

It is an object of the present invention to provide an applicationmethod for marker assisted selection of this bovine gene, and inparticular, of the polymorphisms in the bovine gene which are associatedwith increased milk volume and altered milk composition; and/or toprovide genetic markers for use in such a method; and/or to provide thenucleic acid and amino acid sequences of this gene and encodedpolypeptide; and/or to provide animals selected using the method of theinvention as well as milk produced by the selected animals; and/or toprovide the public with a useful choice.

SUMMARY OF THE INVENTION

This invention relates to the discovery of the bovineDiacylglycerol-o-acyltransferase (DGAT1) gene and polymorphisms withinthe bovine DGAT1 gene which are associated with increased milk yield andaltered milk composition.

More specifically, several polymorphisms- in the bovine DGAT1 gene havebeen identified distinguishing multiple DGAT1 alleles in differentcattle breeds. These polymorphisms include: K232A (Bases 6829/30 AA-CGnucleic acid change and K-A amino acid change); Nt984+8(Base 7438 A-Gnucleic acid change); Nt984+26(Base 7456 C-T nucleic acid change);Nt1470+85(Base 8402 C-T nucleic acid change); Nt191+435 (Base 626 T-Gnucleic acid change); Nt191-3321 (Base 3512 T-G nucleic acid change);Nt279+144 (Base 4040 T-C nucleic acid change); Nt279+1067 (Base 4963 A-Gnucleic acid change); Nt279+1107 (Base 5003 G-A nucleic acid change);Nt358 (Base 5997 C-T nucleic acid change); Nt754+3 (Base 6892 G-Anucleic acid change); Nt897+32 (Base 7224/5 GG-AC nucleic acid change);Nt1251+42 (Base 7987 G-A nucleic acid change) as summarised in Table 1.In particular, DGAT1 alleles characterized by the K232A mutation havebeen identified as being associated with an increased milk volume andaltered milk composition in animals dependent upon whether they arehomozygous with or without the mutation or heterozygous carrying onemutated allele. More specifically, the presence of the K232A mutationresults in a decrease in milkfat percentage, milkfat yield, solid fatcontent and milk protein percentage, while increasing milk volume andmilk protein yield.

The present invention thus relates to the use of the polymorphisms in amethod of identification and selection of a bovine having at least oneof said polymorphisms as well as to providing markers specific for suchidentification. Kits comprising said markers for use in marker selectionalso form part of the present invention as do animals so selected, themilk produced by such selected animals and products produced from suchmilk, particularly as such milk and milk products affect processingand/or health characteristics for consumers.

In particular, the present invention is directed to a method ofgenotyping cows or bulls for one or more of the polymorphisms disclosedherein, selected cows or bulls so genotyped and milk and semen from saidselected cows and bulls respectively.

According to a further aspect the present invention is directed to theisolated DGAT1 nucleic acid and allelic nucleic acid moleculescomprising polymorphisms as well as to the proteins encoded thereby andtheir polypeptide sequences. Antibodies raised against said proteins arealso contemplated, as are vectors comprising the nucleic acid molecules,host cells comprising the vectors; and protein molecules expressed insaid host cells; and the application of all of them in the farmingindustry.

In particular, such applications include methods for modulating milkproduction and/or composition in a lactating bovine by affecting DGAT1activity, by reducing the activity of DGAT1 (e.g. by use of specificribozymes, antisense sequences and/or antibodies, or by transgenictechnology to produce a “knock out” bovine and/or bovine with introducedtransgenes containing the DGAT1 gene and/or variations of this genedriven by various promoters).

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described with reference to the Figures of theaccompanying drawings in which:

FIG. 1: shows a BAC contig spanning the BULGE12-BULGE 09 intervalrelative to a schematic diagram of bovine chromosome 14 and a schematicdiagram showing the location of the genetic markers. The most likelyposition of the QTL is shown as a bar on the FISH-ancored linkage mapproximal to BTA14q. The BACs composing the contigs spanningBULGE13-BULGE09 interval are shown as a series of horizontal lines. Thesymbols on each BAC indicate their individual STS content: solid boxescorrespond to STS derived from BAC ends, open boxes to microsatellitemarkers, and solid triangles to gene-specific Comparative AnchoredTagged Sequences. The arrow heads mark the BACs from which therespective BAC end STS were derived. The length of the lines do notreflect the actual insert size of the corresponding BACs. The BAC contigwas aligned with the orthologous human HSA8q24.3 genomic “golden path”sequence represented according to the Ensembl Human Genome Serverindividual sequence contigs are shown in alternating light and dark; ahorizontal line indicates a gap in the sequence assembly; geneticmarkers are indicated under the contig map; the lines and boxes abovethe contig map represent “curated”, “predicted known” or “predictednovel” genes.

FIGS. 2 a and 2 b: Show the genomic sequence of the bovine DGAT1 gene.FIG. 2 a is the 31 base pair sequence upstream but adjacent to the ATGor translation start site and is 5′ UTR. FIG. 2 b is the genomicsequence in the bovine DGAT1 gene from the ATG translation start site(base 1) through to genomic sequence flanking the gene at the 3′ end.The significant features including intron/exon boundaries, polymorphicsites, polyadenylation signal, and alternate splicing site and some ofthe primer sequences used in the assays described herein, are indicated;

FIG. 3: Shows the genomic organization, four polymorphisms andhaplotypes found in the bovine DGAT1 gene. Leader and trailer sequencesare shown in light grey, coding sequences in dark grey and intronicsequences as a hollow line. The positions of four of the identifiedpolymorphisms are marked as shown on the gene, and detailed in theunderlying boxes including the corresponding sequence traces All thesequence variations are summarised in Table 1. The four DGAT1 haplotypeswhich were found in the Dutch and New-Zealand Holstein-Friesianpopulation as defined by these polymorphisms are shown and referred toas “sH^(Q-D)”, “sH^(Q-NZ)”, “sH^(Q-III)” for the fat increasinghaplotypes and “sh^(q)” for the fat decreasing haplotype;

FIG. 4 a: Shows the corresponding full length amino acid sequence forDGAT1 sequence of FIG. 2 b including annotation of the amino acidsubstitution;

FIG. 4 b: Shows the amino acid sequence predicted as a result ofalternate splicing with exon VIII;

FIG. 5: Shows the multiple peptide alignment of a portion of the DGAT1protein flanking the K232A substitution from Bos taurus, Bison bison,Ovis aries, Sus scrofa, Homo sapiens, Cercopithecus aethiops, Musmusculus domesticus and Rattus noruegicus showing the evolutionaryconservation of the lysine mutated in the bovine K232A polymorphism;

FIG. 6: A. Shows the frequency distribution of observed DGAT1 SNPhaplotypes in the Dutch and New Zealand Holstein-Friesian dairy cattlepopulations. B-D. Shows the frequency distribution of the combinedmicrosatellite (BULGE09-BULGE11) and SNP DGAT1 haplotypes. The H^(Q-D)and H^(Q-NZ) haplotypes are shown; and

FIG. 7: Shows the lod score due to LD when including (+) or excluding(−) the four DGAT1 polymorphisms shown in FIG. 3 in a combined linkageand LD multipoint maximum likelihood mapping method. The lod scorecorresponds to the log₁₀ of the ratio between the likelihood of the dataassuming LD and linkage between the markers and the likelihood of thedata assuming linkage in the absence of LD. The positions of themicrosatellites and SNP markers utilized in the analysis are shown onthe X-axis, while the position of the DGAT1 SNPs is marked by a redarrow at the top of the figure.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered for the first time that the DGAT1 gene in bovineis associated with the QTL on chromosome 14 which is linked withimproved milk production traits. More particularly, a number of novelpolymorphisms on the DGAT1 gene have been discovered. It is thought thatone or more of these polymorphisms is responsible for these traits.

The method used for isolating genes which cause specific phenotypes isknown as positional candidate cloning. It involves: (i) the chromosomallocalisation of the gene which causes the specific phenotype usinggenetic markers in a linkage analysis; and (ii) the identification ofthe gene which causes the specific phenotype amongst the “candidate”genes known to be located in the corresponding region. Most of the timethese candidate genes are selected from available mapping information inhumans and mice.

The tools required to perform the initial localisation (step (i) above)are microsatellite marker maps, which are available for livestockspecies and are found in the public domain (Bishop et al., 1994;Barendse et al., 1994; Georges et al., 1995; and Kappes, 1997). Thetools required for the positional candidate cloning, particularly theBAC libraries, (step (ii) above) are partially available from the publicdomain. Genomic libraries with large inserts constructed with BacterialArtificial Chromosomes (BAC) are available in the public domain for mostlivestock species including cattle. For general principles of positionalcandidate cloning, see Collins, 1995 and Georges and Anderson, 1996.

Recently, a quantitative trait locus (QTL) with major effect on milksolids composition, located at the centromeric end of bovine chromosome14, has been reported (Coppieters et al., (1998)). This QTL was shown toeffect milk fat content and in particular to significantly affectprotein %, volume, protein yield and fat yield of milk. The linkagestudy as well as subsequent marker assisted segregation analyses allowedfor the identification of thirteen Holstein-Friesian sires predicted tobe heterozygous “Qq” for the corresponding QTL (Coppieters et al.,(1998); Riquet et al., (1999)).

Linkage disequilibrium methods were applied to refine the map positionof the QTL to a ≈5 cM interval bounded by microsatellite markers BULGE09and BULGE30.

A bovine DGAT1 nucleotide sequence was determined by the applicants andis shown in FIGS. 2 a and 2 b with the corresponding amino acidsequences (long and short forms) being shown in FIGS. 4 a and 4 brespectively. Table 1 sets out all the polymorphisms located to datewith reference to the sequence in FIG. 2 b. Some of the geneticpolymorphisms identified in the bovine DGAT1 gene are reported in FIG.3. The nucleic acid and protein sequences of the DGAT1 alleles includingthe K232A mutation are shown in FIGS. 2 a and 2 b (SEQ ID NOs: 3 and 1),annotated to show the alternatively spliced forms. The cDNA sequence isalso set out in SEQ ID NO: 4.

The sequence information in the Figures gives rise to numerous, andseparate, aspects of the invention.

In one aspect, the invention provides a method of determining geneticmerit of a bovine with respect to milk composition and volume whichcomprises the step of determining the bovine DGAT1 genotyping state ofsaid bovine. In particular, this method is useful for genotyping andselecting cows and bulls having the desired genotypic state so that milkand semen may be collected from said cows and bulls respectively. Suchsemen would be useful for breeding purposes to produce bovine having thedesired genotypic and, as a result, phenotypic state. In addition, cowsgenotyped by the methods of the present invention are also useful forbreeding purposes, particularly for breeding with the selected bullsand/or to be artificially inseminated with the semen from selectedbulls. The embryos and offspring produced by such cows also form part ofthe present invention.

In one embodiment, the genotypic state is determined with respect to DNAobtained from said bovine.

Alternatively, said genotypic state is determined with reference to mRNAobtained from said bovine.

In yet a further embodiment, the genotypic state is determined withreference to the amino acid sequence of expressed bovine DGAT1 proteinobtained from said bovine.

Conveniently, in said method, the genotypic state of DNA encoding bovineDGAT1 is determined, directly or indirectly.

Alternatively, in said method the genotypic state of at least onenucleotide difference from the nucleotide sequence encoding bovine DGAT1is determined, directly or indirectly.

More specifically, in said method the genotypic state of bovine DGAT1allele(s) characterised by one or more of the polymorphisms shown inTable 1 below, is determined, directly or indirectly.

TABLE 1 Table of polymorphisms in the bovine DGAT1 gene Start codon(atg); the a residue is denoted as position 1 Base number relative toNucleotide distance Intron/exon exonic sequence¹ from start substitutionSEQ ID NO: # Nt 191 + 435 626 T-G Intron 1 CAGTGCTAGGGG 22 CAGTGCGAGGGG23 Nt 191 + 3321 3512 T-G Intron 1 GCATTGCGCT 24 GCATGGCGCT 25 Nt 279 +144 4040 T-C Intron 2 TACCCTGGGAC 26 TACCCCGGGAC 27 Nt 279 + 1067 4963A-G Intron 2 CTCTTAGCAGC 28 CTCTTGGCAGC 29 Nt 279 + 1107 5003 G-A Intron2 ACAGGCAACT 30 ACAGACAACT 31 Nt 358 5997 C-T Exon IV TGTCTCTGTTC 32TGTCTTTGTTC 33 Nt 692 6829 AA-GC K232A Exon GGTAAGAAGGCCAA 34 VIII* (Q)GGTAAGGCGGCCAA 35 (q) Nt 754 + 3 6892 G-A Intron VIII GCGGTGAGGAT 36GCGGTAAGGAT 37 Nt 897 + 32 7224 GG-AC Intron X GGGGGGGGGGGA 38 CTCTGGGGGACGGGGA 39 CTCT Nt 984 + 8 7438 A-G Intron XII* GAGTGACCTGC 40GAGTGGCCTGC 41 Nt 984 + 26 7456 C-T Intron XII* GGACGCGTGGG 42GGACGTGTGGG 43 Nt 1251 + 42 7987 G-A Intron XV GGTGGGGGTGG 44GGTGGAGGTGG 45 Nt 1470 + 85 8402 C-T 3′ flanking CTGGGCGCAGC 46 region *CTGGGTGCAGC 47 The numbers given are far the actual nucleotide or in thecase of two nucleotide substitutions to the first nucleotide in thevariation (counting 5′ to 3′ *More detail of these polymorphisms isgiven in FIG. 2b. ¹e.g. Nt 191 represents nucletode number 191 from thestart site of the coding sequence, + 435 represents number ofnucleotides from and including base 192 in the genomic sequence(intron 1) to the polymorphic nucleotide The polymorphic nucleotides areshadedPreferably, the invention is directed to a method of determining thegenotypic state of bovine DGAT1 allele(s) by determining the presence ofthe K232A polymorphism, either directly or indirectly.

There are numerous art standard methods known for determining whether aparticular DNA sequence is present in a sample. An example is thePolymerase Chain Reaction (PCR). A preferred aspect of the inventionthus includes a step in which ascertaining whether a polymorphism(s) inthe sequence of DGAT1 DNA is present, includes amplifying the DNA in thepresence of primers based on the nucleotide sequence of the DGAT1 geneand flanking sequence, and/or in the presence of a primer containing atleast a portion of a polymorphism as disclosed herein and which whenpresent results in altered relative milk lipid and protein production,and milk volume.

A primer of the present invention, used in PCR for example, is a nucleicacid molecule sufficiently complementary to the sequence on which it isbased and of sufficient length to selectively hybridise to thecorresponding portion of a nucleic acid molecule intended to beamplified and to prime synthesis thereof under in vitro conditionscommonly used in PCR. Likewise, a probe of the present invention, is amolecule, for example a nucleic acid molecule of sufficient length andsufficiently complementary to the nucleic acid molecule of interest,which selectively binds under high or low stringency conditions with thenucleic acid sequence of interest for detection thereof in the presenceof nucleic acid molecules having differing sequences.

In another aspect, the invention provides a method for determining thegenetic merit of bovine with respect to milk content and volume withreference to a sample of material containing mRNA obtained from thebovine. This method includes ascertaining whether a polymorphism(s) inthe sequence of the mRNA encoding DGAT1 is present. The presence of suchpolymorphisms again indicates an association with altered relative milklipid and protein production and milk volume.

Again, if an amplification method such as PCR is used in ascertainingwhether a polymorphism(s) in the sequence of the mRNA encoding (DGAT1)is present, the method includes reverse transcribing the mRNA using areverse transcriptase to generate a cDNA and then amplifying the cDNA inthe presence of a pair of primers complementary to a nucleotide sequenceencoding a protein having biological activity of wild type DGAT1.

In a further aspect, the invention includes the use of a probe in themethods of genotyping according to the invention wherein the probe isselected from any 5 or more contiguous nucleotides of the DGAT1 sequenceas shown in FIG. 2 b, which is therefore sufficiently complementary witha nucleic acid sequence encoding such bovine DGAT1, or its complement,so as to bind thereto under stringent conditions. Diagnostic kitscontaining such a probe are also included. Such probes may be selectedfrom ForAA (FAM): CGTTGGCCTTCTTA or DgatADGC (VIC): TTGGCCGCCTTACC. (SEQID NOs: 20 and 21 respectively.)

The invention further includes isolated nucleic acid molecules encodingthe DGAT1 variant proteins i.e. those proteins encoded by SEQ ID NOs: 1and 4 (FIG. 2 b), comprising one or more polymorphisms of SEQ ID NOs. 7to 19 (Table 1), or a fragment or variant thereof. Particularly, theinvention includes an isolated nucleic acid molecule comprising a DNAmolecule having in whole or in part the nucleotide sequence identifiedin FIG. 2 b or which varies from the sequence due to the degeneracy ofthe genetic code, or a nucleic acid strand capable of hybridising withsaid nucleic acid molecule under stringent hybridisation conditions.

The invention includes isolated mRNA transcribed from DNA having asequence which corresponds to a nucleic acid molecule of the invention.

The invention includes isolated DNA in a recombinant cloning vector anda prokaryotic or eukaryotic cell containing and expressing heterologousDNA of the invention.

The invention includes a transfected coil line which expreses a proteinencoded by the nucleic acid molecules of the invention.

The invention also includes a primer composition useful for detection ofthe presence of one or more polymorphisms associated with improved milkproduction traits in bovine DNA encoding DGAT1 and/or the presence ofDNA encoding a variant protein. In one form, the composition can includea nucleic acid primer substantially complementary to a nucleic acidsequence encoding DGAT1. The nucleic acid sequence can in whole or inpart be that identified in FIG. 2 b. Diagnostic kits including such acomposition are also included.

The invention further provides a diagnostic kit useful in detecting DNAencoding a variant DGAT1 protein in bovine which includes first andsecond primers for amplifying the DNA, the primers being complementaryto nucleotide sequences of the DNA upstream and downstream,respectively, of a polymorphism in the portion of the DNA encoding DGAT1which results in altered relative milk lipid, solid fat content andprotein production and milk volume, wherein at least one of thenucleotide sequences is elected to be from a non-coding region of theDGAT1 gene. The kit can also include a third primer complementary to apolymorphism, disclosed herein, located on the DGAT1 gene.

The invention includes a process for producing a protein of theinvention, including preparing a DNA fragment including a nucleotidesequence which encodes the protein; incorporating the DNA fragment intoan expression vector to obtain a recombinant DNA molecule which includesthe DNA fragment and is capable of undergoing replication; transforminga host cell with the recombinant DNA molecule to produce a transformantwhich can express the protein; culturing the transformant to produce theprotein; and recovering the protein from resulting cultured mixture.

Thus in a further aspect, the invention provides a purified proteinencoded by the nucleic acid molecule of the invention and havingbiological activity of DGAT1. The terms “isolated” and “purified” asused herein, each refer to a protein substantially free of cellularmaterial or culture medium when produced by recombinant DNA techniques,or chemical precursors or other chemicals when chemically synthesised.In certain preferred embodiments, the protein having biological activityof DGAT1 comprises an amino acid sequence and variants shown in FIGS. 4a and 4 b (SEQ ID NOs: 2, 5 and 6). Furthermore, proteins havingbiological activity of DGAT1 that are encoded by nucleic acids whichhybridise under stringent conditions to a nucleic acid comprising anucleotide sequence shown in FIG. 2 b (SEQ ID NOs: 1 and 4) areencompassed by the invention.

Proteins of the invention having DGAT1 activity can be obtained byexpression of a nucleic acid coding sequence in a suitable host cellusing techniques known in the art. Suitable host cells includeprokaryotic or eukaryotic organisms or cell lines, for example, yeast,E. coli, insect cells and COS 1 cells. The recombinant expressionvectors of the invention can be used to express a protein having DGAT1activity in a host cell in order to isolate the protein. The inventionprovides a method of preparing a purified protein of the inventioncomprising introducing into a host cell a recombinant nucleic acidencoding the protein, allowing the protein to be expressed in the hostcell and isolating and purifying the protein. Preferably, therecombinant nucleic acid is a recombinant expression vector. Proteinscan be isolated from a host cell expressing the protein and purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, column chromatography (eg. ion exchange, gel filtration,affinity chromatography, etc.) electrophoresis, and ultimately,crystallisation (see generally “Enzyme Purification and RelatedTechniques”. Methods in Enzymology, 22, 233-577 (1971)).

Alternatively, the protein or parts thereof can be prepared by chemicalsynthesis using techniques well known in the chemistry or proteins suchas solid phase synthesis (Merrifield, 1964), or synthesis in homogeneoussolution (Houbenwcyl, 1987).

It will of course be understood that a variety of substitutions of aminoacids is possible while preserving the structure responsible foractivity of the DGAT1 proteins disclosed herein. Conservativesubstitutions are described in the patent literature, as for example, inU.S. Pat. No. 5,264,558 or 5,487,983. It is thus expected, for example,that interchange among non-polar aliphatic neutral amino acids, glycine,alanine, proline, valine and isoleucine, would be possible. Likewise,substitutions among the polar aliphatic neutral amino acids, serine,threonine, methionine, asparagine and glutamine could possibly be made.Substitutions among the charged acidic amino acids, aspartic acid andglutamic acid, could probably be made, as could substitutions among thecharged basic amino acids, lysine and arginine. Substitutions among thearomatic amino acids, including phenylalanine, histidine, tryptophan andtyrosine would also likely be possible. These sorts of substitutions andinterchanges are well known to those skilled in the art. Othersubstitutions might well be possible. Of course, it would also beexpected that the greater percentage of homology ie. sequencesimilarity, of a variant protein with a naturally occurring protein, thegreater the retention of activity.

A further advantage may be obtained through chimeric forms of theproteins, as known in the art. A DNA sequence encoding each entireprotein, or a portion of the protein, could be linked, for example, witha sequence coding for the C-terminal portion of E. coli β-galactosidaseto produce a fusion protein.

The proteins of the invention, or portions thereof, have numerousapplications in turn. By way of example, each protein can be used toprepare antibodies which bind to a distinct epitope in an unconservedregion of the protein. An unconserved region of the protein is one whichdoes not have substantial sequence homology to other proteins.

Still further, the invention includes an antibody to a bovine DGAT1variant protein encoded by a nucleotide sequence of the presentinvention as well as a diagnostic kit containing such an antibody.

Conventional methods can be used to prepare the antibodies.. Forexample, by using a DGAT1 peptide, polyclonal antisera or monoclonalantibodies can be made using standard methods. A mammal, (eg. a mouse,hamster, or rabbit) can be immunised with an immunogenic form of thepeptide which elicits an antibody response in the mammal. Techniques forconferring immunogenicity on a peptide include conjugation to carriersor other techniques well known in the art. For example, the peptide canbe administered in the presence of adjuvant. The progress ofimmunisation can be monitored by detection of antibody titers in plasmaor serum. Standard ELISA or other immunoassay can be used to assess thelevels of antibodies. Following immunisation, antisera can be obtainedand, if desired, polyclonal antibodies isolated from the sera.

To produce monoclonal antibodies, antibody producing cells (lymphocytes)can be harvested from an immunised animal and fused with myeloma cellsby standard somatic cell fusion procedures, thus immortalising thesecells and yielding hybridoma cells. Such techniques are well known inthe art. For example, the hybridoma technique originally developed byKohler and Milstein (Kohler, 1975) as well as other techniques such asthe human B-cell hybridoma technique (Kozbor, 1983) and screening ofcombinatorial antibody libraries (Huse, 1989). Hybridoma cells can bescreened immunochemically for production of antibodies specificallyreactive with the peptide, and monoclonal antibodies isolated.

The term “antibody” as used herein is intended to include fragmentsthereof which are also specifically reactive with the target protein.Antibodies can be fragmented using conventional techniques and thefragments screened for utility in the same manner as described above forwhole antibodies. For example, F(ab′)₂ fragments can be generated bytreating antibody with pepsin. The resulting F(ab′)₂ fragment can betreated to reduce disulfide bridges to produce Fab′ fragments.

Another method of generating specific antibodies, or antibody fragments,reactive against the target proteins is to screen expression librariesencoding immunoglobulin genes, or portions thereof, expressed inbacteria, with peptides produced from the nucleic acid molecules of thepresent invention. For example, complete Fab fragments, VH regions andFV regions can be expressed in bacteria using phage expressionlibraries. See for example Ward et al., Huse et al., and McCafferty etal. (Ward, 1989); Huse 1989; McCafferty, 1990). Screening such librarieswith, for example, a DGAT1 protein can identify immunoglobulin fragmentsreactive with that DGAT1. Alternatively, the SCID-hu mouse developed byGenpharm can be used to produce antibodies, or fragments thereof.

The polyclonal, monoclonal or chimeric monoclonal antibodies can be usedto detect the proteins of the invention, portions thereof or closelyrelated isoforms in various biological materials. For example, they canbe used in an ELISA, radioimmunoassay or histochemical tests. Thus, theantibodies can be used to quantify the amount and location of a DGAT1protein of the invention, portions thereof or closely related isoformsin a sample in order to determine the role of DGAT1 proteins. Usingmethods described hereinbefore, polyclonal, monoclonal antibodies, orchimeric monoclonal antibodies can be raised to non-conserved regions ofDGAT1 and used to distinguish a particular DGAT1 from other proteins.

The polyclonal or monoclonal antibodies can be coupled to a detectablesubstance or reporter system. The term “coupled” is used to mean thatthe detectable substance is physically linked to the antibody. Suitabledetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials and radioactive materials.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, and acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin;an example of a luminescent material includes luminol; and examples ofsuitable radioactive material include ¹²⁵I; ¹³¹I, ³⁵S and ³H. In apreferred embodiment, the reporter system allows quantitation of theamount of protein (antigen) present.

Such an antibody-linked reported system could be used in a method fordetermining whether a fluid or tissue sample of a bovine contains adeficient amount or an excessive amount of the relevant DGAT1 protein.Given a normal threshold concentration of such a protein, test kits canbe developed.

The availability of such antibodies gives rise to further applications.One is a diagnostic kit for identifying cells comprising an antibody(such as a monoclonal antibody) which binds to a protein comprising anamino acid sequence shown in FIG. 4 a and 4 b; means for detecting theantibody when bound to the protein, unreacted protein or unboundantibody; means for determining the amount of protein in the sample; andmeans for comparing the amount of protein in the sample with a standard.In some embodiments of the invention, the detectability of the antibodywhich binds to a specific DGAT1 protein is activated by the binding (eg.change in fluorescence spectrum, loss of radioisotopic label). Thediagnostic kit can also contain an instruction manual for use of thekit.

Antibody-based diagnostics are of course not the only possibility. Afurther diagnostic kit comprises a nucleotide probe complementary to thesequence, or an oligonucleotide fragment thereof, shown in FIG. 2 a and2 b, for example, for hybridisation with mRNA from a sample of cells;means for detecting the nucleotide probe bound to mRNA in the samplewith a standard. In a particular aspect, the kit of this aspect of theinvention includes a probe having a nucleic acid molecule sufficientlycomplementary with a sequence identified in FIG. 2 a and 2 b, or itscomplement, so as to bind thereto under stringent conditions. “Stringenthybridisation conditions” takes on its common meaning to a personskilled in the art. Appropriate stringency conditions which promotenucleic acid hybridisation, for example, 6×sodium chloride/sodiumcitrate (SSC) at about 45° C. are known to those skilled in the art,including in Current Protocols in Molecular Biology, John Wiley & Sons,N.Y. (1989). Appropriate wash stringency depends on degree of homologyand length of probe. If homology is 100%, a high temperature (65° C. to75° C.) may be used. If homology is low, lower wash temperatures must beused. However, if the probe is very short (<100bp), lower temperaturesmust be used even with 100% homology. In general, one starts washing atlow temperatures (37° C. to 40° C.), and raises the temperature by 3-5°C. intervals until background is low enough not to be a major factor inautoradiography. The diagnostic kit can also contain an instructionmanual for use of the kit.

One of the major applications of the present invention is in the markerassisted selection of bovines having a polymorphism in the DGAT1 geneand which are associated with improved milk production traits. Theinvention therefore provides a diagnostic kit which can be used todetermine the DGAT1 genotype of bovine genetic material, for example.One kit includes a set of primers used for amplifying the geneticmaterial. A kit can contain a primer including a nucleotide sequence foramplifyg a region of the genetic material containing one of thepolymorphisms described herein. Such a kit could also include a primerfor amplifying the corresponding region of the normal DGAT1 gene, i.e.the sequence without polymorphisms. Usually, such a kit would alsoinclude another primer upstream or downstream of the region of interestcomplementary to a coding and/or non-coding portion of the gene. Theseprimers are used to amplify the segment containing the mutation, i.e.polymorphism, of interest.

In particular, the invention is directed to the use of the polymorphismsin the DGAT1 gene in the genotyping of cows and bulls as well as to cowsand bulls selected by such genotyping which have one or more of saidpolymorphisms in the DGAT1 gene. Such bulls so selected are of valuablebreeding stock and the invention is also directed to the semen producedby such selected bulls for breeding purposes. Cows so selected are alsouseful as breeding stock as are their offspring. In addition, such cowsmay produce valuable dairy herds as the milk produced by such cows isproduced in greater volumes than equivalent non-selected cows, and/orhas an altered composition in that it comprises less milkfat and moremilk protein. Such milk and products made therefrom also form part ofthe invention. It is also noted that the milk from these selected cowswill be valuable as the fat content is not only decreased but is alsocharacterised by being softer. Without being bound by theory, it isthought that this increased fat softness is due to the fatty acidcomposition being such that there is less saturated and more unsaturatedfat in the milk of selected cows. Thus it is anticipated that productsmade from such milk will have processing advantages, such as in theproduction of more spreadable butter, as well as having a health benefiton consumers, as generally unsaturated fats are considered to be more“healthy” than saturated fats. The protein composition of milk producedby such selected cows is also altered. In particular, such milkcomprises an altered protein yield compared to milk for nonselected cowsand the casein:whey ratio is also altered which makes such milk valuablefor cheese production.

Thus, the present invention involves genotyping bovine, both cows andbulls, for the DGAT1 polymorphisms disclosed herein, selected cows andbulls so genotyped, milk and semen produced by the selected cows andbulls so genotyped, offspring produced by the selected bovine, includingembryos and cells (including cell lines) useful for cloning saidselected bovine.

The actual genotyping is carried out using primers that target specificpolymorphisms as described herein and that could function asallele-specific oligonucleotides in conventional hybridisation, Taqmanassays, OLA assays, etc. Alternatively, primers can be designed topermit genotyping by microsequencing.

One kit of primers can include first, second and third primers, (a), (b)and (c), respectively. Primer (a) is based on a region containing aDGAT1 mutation such as described above. Primer (b) encodes a regionupstream or downstream of the region to be amplified by primer (a) sothat genetic material containing the mutation is amplified, by PCR, forexample, in the presence of the two primers. Primer (c) is based on theregion corresponding to that on which primer (a) is based, but lackingthe mutation. Thus, genetic material containing the non-mutated regionwill be amplified in the presence of primers (b) and (c). Geneticmaterial homozygous for the DGAT1 gene will thus provide amplifiedproducts in the presence of primers (b) and (c). Genetic materialhomozygous for the mutated gene will thus provide amplified products inthe presence of primers (a) and (b). Heterozygous genetic material willprovide amplified products in both cases.

The present invention also contemplates the modulation of milkproduction and content in non-human animals by modulating the activityof the DGAT1 protein. In particular, this aspect of the inventionincludes a method of modulating milk production and/or milk content in alactating bovine, the method comprising administering to the bovine aneffective amount of a nucleic acid molecule substantially complementaryto at least a portion of mRNA encoding the bovine DGAT1 variant proteinsand being of sufficient length to sufficiently reduce expression of saidDGAT1, i.e. by use of antisense nucleic acids.

Antisense nucleic acids or oligonucleotides (RNA or preferably DNA) canbe used to inhibit DGAT1 production in a bovine if this is considereddesirable e.g. in order to produce a bovine capable of improved milkproduction, i.e. increased milk volume and decreased milkfat content.Antisense oligonucleotides, typically 15 to 20 bases long, bind to thesense mRNA or pre mRNA region coding for the protein of interest, whichcan inhibit translation of the bound mRNA to protein. The cDNA sequenceencoding DGAT1 can thus be used to design a series of oligonucleotideswhich together span a large portion, or even the entire cDNA sequence.These oligonucleotides can be tested to determine which provides thegreatest inhibitory effect on the expression of the protein (Stewart1996). The most suitable mRNA target sites include 5′- and3′-untranslated regions as well as the initiation codon. Other regionsmight be found to be more or less effective.

Alternatively, an antisense nucleic acid or oligonucleotide may bind toDGAT1 coding sequences.

In yet another embodiment, the invention provides a method of modulatingmilk production and/or milk content in a lactating bovine, includingadministering to the bovine an effective amount of a nucleic acidmolecule having ribozyme activity and a nucleotide sequencesubstantially complementary to at least a portion of MRNA encoding abovine DGAT1 and being of sufficient length to bind selectively theretoto sufficiently reduce expression of said DGAT1.

Rather than reducing DGAT1 activity in the bovine by inhibiting geneexpression at the nucleic acid level, activity of the relevant DGAT1protein may be directly inhibited by binding to an agent, such as, forexample, a suitable small molecule or a monoclonal antibody.

Thus, the invention also includes a method of inhibiting the activity ofbovine DGAT1 in a lactating bovine so as to modulate milk productionand/or milk solids content, comprising administering an effective amountof an antibody to the relevant DGAT1.

The invention still further includes a method of modulating milkproduction and/or milk solids content by raising an autoantibody to abovine DGAT1 in the bovine. Raising the autoantibody can includeadministering a protein having DGAT1 activity to the bovine.

In still a further embodiment, nucleic acids which encode DGAT1 proteinscan be used to generate transgenic animals. A transgenic animal (eg. amouse) is an animal having cells that contain a transgene, whichtransgene is introduced into the animal or an ancestor of the animal ata prenatal, eg. an embryonic stage. A transgene is DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops. In one embodiment, a bovine cDNA, comprising the nucleotidesequence shown in FIG. 2 b, or an appropriate variant or subsequencethereof, can be used to generate transgenic animals that contain cellswhich express the relevant DGAT1. Likewise, variants can be used togenerate transgenic animals. “Knock out” animals can also be generated.

Methods for generating transgenic animals, particularly animals such asmice, have become conventional in the art are described, for example, inU.S. Pat. Nos. 4,736,866 and 4,870,009. In such methods, plasmidscontaining recombinant molecules are microinjected into mouse embryos.In particular, the plasmids can be microinjected into the male pronucleiof fertilised one-cell mouse eggs; the injected eggs transferred topseudo-pregnant foster females; and the eggs in the foster femalesallowed to develop to term. (Hogan, 1986). Alternatively, an embryonalstem cell can be transfected with an expression vector comprisingnucleic acid encoding a DGAT1 protein, and cells containing the nucleicacid can be used to form aggregation chimeras with embryos from asuitable recipient mouse strain. The chimeric embryos can then beimplanted into a suitable pseudopregnant female mouse of the appropriatestrain and the embryo brought to term. Progeny harbouring thetransfected DNA in their germ cells can be used to breed uniformlytransgenic mice.

Such animals could be used to determine whether a sequence related to anintact DGAT1 gene retains biological activity of the encoded DGAT1.Thus, for example, mice in which the murine DGAT1 gene has been knockedout and containing the nucleic acid sequence identified in FIG. 2 b orfragment or variant thereof could be generated. The animals could beexamined with reference to milk production and content.

The pattern and extent of expression of a recombinant molecule of theinvention in a transgenic mouse is facilitated by fusing a reporter geneto the recombinant molecule such that both genes are co-transcribed toform a polycistronic MRNA. The reporter gene can be introduced into therecombinant molecule using conventional methods such as those describedin Sambrook et al., (Sambrook, 1989). efficient expression of bothcistrons of the polycistronic mRNA encoding the protein of the inventionand the reporter protein can be achieved by inclusion of a knowninternal translational initiation sequence such as that present inpolivirus mRNA. The reported gene should be under the control of theregulatory sequence of the recombinant molecule of the invention and thepattern and extent of expression of the gene encoding a protein of theinvention can Accordingly be determined by assaying for the phenotype ofthe reporter gene. Preferably the reporter gene codes for a phenotypenot displayed by the host cell and the phenotype can be assayedquantitatively Examples of suitable reporter genes include lacZ(β-galactosidase), neo (neomycin phosphotransferase), CAT(chloramphenicol acetyltransferase) dhfr (dihydrofolate reductase),aphIV (hygromycin phosphotransferase), lux (luciferase), uidA(β-glucuronidase), Preferably, the reporter gene is lacZ which codes forβ-galactosidase. β-galactosidase can be assayed using the lactoseanalogue X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) whichis broken down by β-galactosidase to a product that is blue in colour.

Still further transgenic applications of the invention arise fromknocking out the endogenous gene encoding DGAT1 in non-human mammals andreplacing this with a bovine transgene, in order to obtain a desiredeffect. This is particularly true in cattle raised for milk production.For example, additional copies of the bovine gene encoding DGAT1 can beinserted as a transgene, or the endogenous gene associated with a highlevel expression promoter in a transgene. It may also prove advantageousto substitute a defective gene rather than delete the entire sequence ofDNA encoding for a protein having DGAT1 activity. A method of producinga transgenic bovine or transgenic bovine embryo is described in U.S.Pat. No. 5,633,076, issued May 27, 1997, for example.

These transgenic animals of the invention can again be used toinvestigate the molecular basis of DGAT1 action. For example, it isexpected that mutants in which one or more of the conserved cysteineresidues has been deleted would have diminished activity in relation toa DGAT1 protein in which all such residues are retained. Further,deletion of a proteolytic cleavage site would likely result in a mutantlacking biological activity of DGAT1.

Transgenic animals of the invention can also be used to test substancesfor the ability to prevent, slow or enhance DGAT1 activity. A transgenicanimal can be treated with the substance in parallel with an untreatedcontrol transgenic animal. Substances which could be tested in this wayinclude proteins extracted from foods ingested by the animal, Forexample, proteins extracted from pastoral grasses and other fodder canbe tested to determine their effect on DGAT1 activity, including todetermine Whether breed-specific effects can be induced.

Thus, in further aspects, the invention provides transgenic non-humananimals. These include by way of example only a transgenic bovine havinga genome lacking a gene encoding a protein having biological activity ofDGAT1 (or indeed any DGAT1 activity at all); a transgenic mouse having agenome containing a gene encoding a bovine protein having biologicalactivity of any DGAT1; and a transgenic bovine having a gene, encoding abovine protein having biological activity of a bovine DGAT1 andheterologous nucleotide sequence antisense to the gene. The transgenicbovine can include a gene encoding a nucleic acid sequence havingribozyme activity and in transcriptional association with the nucleotidesequence antisense to the gene.

The invention further provides a transgenic bovine having a genome whichincludes additional copies of a gene encoding a protein havingbiological activity of DGAT1 or copies of a gene encoding a proteinhaving biological activity of DGAT1 under control of a high expressionpromoter.

These are but a selection of the applications of this invention. Otherswill be apparent to those persons skilled in this art and are in no wayexcluded. To the contrary, the invention extends to cover not only thespecific teaching provided but also all variations and modificationswhich are within the skill and contemplation of the addressee.

The invention will now be defined by specific examples which areillustrative only and are not intended to limit the invention in anyway.

Experimental

1. Location of the Gene Responsible for the Observed QTL

Construction of a BAC contig Spanning the BULGE9-BULGE30 Interval.

In order to clone the gene(s) responsible for the observed QTL effect, aBAC contig spanning the corresponding marker interval was constructed.This was accomplished by screening a BAC library by filter hybridisationwith the microsatellite markers available for proximal BTA14q, as wellas with human cDNA clones mapping to the orthologous chromosome segmenton the human RH transcript map: 8q23.3-ter (Riquet et al., (1999)). Theends of the isolated BACs were sequenced, sequence tagged sites (STS)developed from the corresponding sequences, and mapped onto a bovine xhamster whole genome radiation hybrid panel. This STS content mappingapproach lead to the construction of the BAC contig shown in FIG. 1.

DGAT1 maps to the BULGE9-BULGE30 Interval and is a Strong PositionalCandidate for the QTL.

A murine gene encoding a protein with Diacylglycerol-o-acyltransferase(DGAT1) activity was identified (Cases et aL, (1998)) and shown tocompletely inhibit lactation when knocked out in the mouse (Smith etal., (2000)). This gene was reported in the human to map to HSA8qter(Cases et al., (1998)), ie. in the region orthologous to that containingthe bovine QTL. Screening the publicly available databases with thepublished murine and human DGAT1 cDNA sequences allowed identificationof (i) a human BAC clone containing the human DGAT1 gene (AF205589), and(ii) three bovine Expressed Sequence Tags (AW446908; AW446985; AW652329)jointly covering approximately two thirds of the bovine gene. Aligningthe human DGAT1 genomic sequences with the human and bovine cDNAsequences allowed the corresponding intron-exon boundaries to beidentified. Primers were developed to PCR amplify a portion of thebovine DGAT1 gene. Screening the BACs composing the BULGE9-BULGE30contig clearly indicated that the bovine DGAT1 gene was contained in asubset of the BACs allowing us to accurately position the DGAT1 gene inthe contig of FIG. 1.

These results demonstrated that the map position of DGAT1 coincided withthe most likely position of the chromosome 14 QTL as determined bylinkage and linkage disequilibrium analyses. Knowing that the QTLprimarily affects fat content, knowing the enzymatic activity of DGAT1and the effect of a DGAT1 knock-out on lactation, this gene wasconsidered to be a very strong positional candidate for thecorresponding QTL.

Organisation of the Bovine DGAT1 Gene

The organisation of the bovine DGAT1 gene was determined by sequenceanalysis of one of the DGAT1 containing BACs. Primers were designedbased on the available bovine, murine and human cDNA sequences whichwere either used for direct sequencing of the BAC clone or to generatePCR products corresponding to different parts of the bovine DGAT1 genefrom this BAC which were then subjected to cycle-sequencing. Allavailable sequences were then merged using the Phred/Phrap software(Ewing et al., (1998); Ewing & Green, (1998); Gordon et al., (1998)) toyield the consensus sequence shown in FIGS. 2 a and b.

RT-PCR, 5′ and 3′ RACE experiments were performed on mRNA isolated frombovine mammary gland and the obtained PCR products subjected to cyclesequencing. Comparison of the genomic and cDNA sequences showed that thebovine DGAT1 gene spans 8.6 Kb and comprises 17 exons measuring 121.8 bpon average (range: 42 -436 bp) and allowed intron-exon boundaries to beidentified (FIGS. 2 a, 2 b and 3). The cDNA sequence is also set out inSEQ ID NO: 4. While the first two introns are respectively 3.6 and 1.9Kb long, the remaining 14 introns are only 92.4 bp long on average(range: 70 -215 bp). All introns conform to the GT-AG rule and arestrictly conserved between human and bovine. The bovine DGAT1 gene istranscribed in a mRNA comprising >31 bp of 5′ UTR sequence (FIG. 2 a),1470 bp coding for a protein of 489 amino-acids, and 275 bp of 3′ UTRsequence including a canonical AATAAA polyadenylation signal. The humanand bovine DGAT1 nucleotide (coding) and protein sequences arerespectively 89.5% and 92.5% identical (FIGS. 2 a, 2 b, 4 a and 4 b). Inaddition, an alternative splicing variant is predicted in the bovine forexon VIII (FIG. 2 b). The corresponding bovine cDNAs are predicted toencode proteins comprising respectively 489 and 467 (alternativesplicing variant) amino-acid residues (FIGS. 4 a and 4 b).

The Predicted “Q” and “q” QTL Alleles Differ by a Non ConservativeLysine to Alanine Amino-acid Substitution in the DGAT1 Gene.

Assuming that DGAT1 is indeed the QTL, it is predicted that theidentified “Q” and “q” QTL alleles will correspond to functionallydistinct DGAT1 alleles, ie. will differ at one or more mutations causingthese alleles to be functionally different. To test this hypothesis, thestructure of the DGAT1 gene in individuals predicted to be of differentQTL genotypes: “QQ”, “Qq” and “qq” was examined. More specifically, theDGAT 1 gene from:

-   -   (i) two sires with “H^(Q-D)/h^(q)” genotype as well as two of        their “H^(Q-D)/H^(Q-D)” offspring, two of their “hq/h^(q)”        offspring and one “H^(Q-D)/h^(q)” offspring, and    -   (ii) one “H^(Q-NZ)/h^(q)” sire with one of its        “H^(Q-NZ)/H^(Q-NZ)” offspring        was analysed wherein H^(Q-D) corresponds to the Dutch Q        haplotype and H^(Q-NZ) corresponds to the New Zealand Q        haplotype, and primer pairs were designed that allowed for the        amplification from genomic DNA of (i) the coding portion of exon        I, (ii) exon II, and (iii) the chromosome regions spanning exons        III to XVII. The corresponding PCR products from the selected        individuals were cycle-sequenced and the resulting sequences        examined with the Polyphred software.

Additional sequencing analysis, as described above, on DNA from a rangeof breeds revealed additional polymorphisms included in Table 1 (seeMethods section for breeds). Four such polymorphisms were investigatedfurther:

(i) K232A: a substitution of a ApA by a GpC dinucleotide in exon VIII(respectively positions 694 and 695 counting from the start codon in thecDNA). The substitution of these two adjacent nucleotides results in anon conservative lysine (hydrophylic basic amino acid) to alanine(hydrophobic amino acid) substitution in the DGAT1 protein. The lysineresidue affected by this polymorphism is conserved in the human andmurine DGAT1 sequences. Together with the resulting change in theelectrical charge of the protein, this strongly suggests that thisamino-acid substitution is likely to result in a functional differencebetween the two corresponding alleles and to be at least partlyresponsible for the observed QTL effect.

(ii) Nt984+8(Base 7438 A-G): A A to G substitution in intron 12, eightbase pairs downstream of exon XII. Following standard nomenclature, thispolymorphism will be referred to as Nt984+8(A-G). This polymorphismcannot be predicted as such to modify the functionality of thecorresponding alleles although an effect on the splicing mechanismcannot be excluded given its proximity to the intron-exon boundary.

(iii) Nt984+26(Base 7456 C-T): A C to T substitution in intron 12, 26base pairs downstream of exon XII. Following standard nomenclature, thispolymorphism will be referred to as Nt984+26(Base 7456 C-T). Again, thispolymorphism cannot be predicted as such to modify the functionality ofthe corresponding alleles although an effect on the splicing mechanismcannot be excluded given its proximity to the intron-exon boundary.

(iv) Nt1470+85(Base 8402 C-T): A C to T substitution in the 3′ UTR.Following standard nomenclature, this polymorphism will be referred toas Nt1470+85(Base 8402 C-T). Again, this polymorphism cannot bepredicted as such to modify the functionality of the correspondingalleles although an effect on polyadenylation or mRNA stability cannotbe excluded.

Conclusion

These four polymorphisms were shown to assort into three distinct SNPhaplotypes referred to as sH^(Q-D), sH^(Q-NZ) and sh^(q) because in thesequenced samples they coincided respectively with microsatellitehaplotypes μH^(Q-D), μH^(Q-NZ) and μh^(q). The base pair compositions ofthese three SNP haplotypes are shown in FIG. 3.

Because the sH^(Q-NZ) and sh^(q) marker haplotypes share the G residueat the DGAT1 Nt984+8(Base 7438 A-G) site, the causality of thispolymorphism in the determinism of the QTL could be excluded. For thethree remaining polymorphic sites, however, the DGAT1 haplotypesassociated with marker haplotypes sHED and sHQIz proved identical toeach other while different from the sh^(q) DGAT1 haplotype. Either ofthese three polymorphisms could therefore be responsible for theobserved QTL effect. The Nt984+26(Base 7456 C-T) and Nt1470+85(Base 8402C-T) polymorphisms are a priori more likely to be neutral with respectto DGAT1 activity because of their respective location in an intron andthe 3′ UTR and likewise the other non coding or neutral polymorphismshown in Table 1. A direct effect of the K232A mutation on DGAT1activity, however, is very plausible. Indeed, the corresponding lysineresidue is conserved amongst all examined mammals (i.e. human, mouse,rat, pig, sheep, bison) demonstrating its functional importance (FIG.5). The evolutionary conservation of this lysine residue alsodemonstrates that the K residue characterizing the sH^(Q-D) andsH^(Q-NZ) marker haplotypes is more than likely the ancestral state andthat it is the A residue characterizing the sh^(q) haplotypes thatcorresponds to a more recently evolved state.

2. Genotype Testing and Analysis I

This summarises the genotype testing and subsequent analysis ofHolstein-Friesian animals sourced from New Zealand and Holland whichwere tested for the presence of the K232A polymorphism. Reference toallele “Q” corresponds to the K residue and allele “q” to the A residue(as shown in FIG. 3 and Table 1).

An oligonucleotide ligation assay (OLA) was developed as described inthe method section below that allows for efficient genotyping of thefour DGAT1 polymorphisms simultaneously. This OLA-test was used togenotype a previously described (Farnir et. al., 2000) “grand-daughterdesign” (i.e. series of paternal half-brother pedigrees) comprising1,818 Dutch Holstein-Friesian sires as well as a “daughter design” (i.e.series of paternal half-sister pedigrees) comprising 529 New ZealandHolstein-Friesian cows selected according to phenotype as describedbelow. The marker linkage phase for each individual was determined asdescribed below.

FIG. 6 summarizes the frequency distribution of DGAT1 haplotypesencountered in the Dutch and New Zealand populations respectively. Fourdistinct SNP haplotypes were identified. Three of these correspond tothe sH^(Q-D), sH^(Q-NZ) and sh^(q) that were previously identified bysequencing, and jointly account for 99% and 98% of the chromosomes inthe Dutch and New-Zealand populations respectively. A fourth minorhaplotype was found accounting for the remaining 1% and 2% of thechromosomes. As this haplotype codes for a K residue at position 232 itwas assumed to correspond to a fat increasing “Q” allele and wastherefore referred to as sH^(Q-III) (FIG. 3). The observation that the Kresidue is found on three distinct DGAT1 haplotypes while the A residueis found on a unique DGAT1 haplotype is in agreement with K being themore ancient state.

The sH_(Q-D) and sH^(Q-NZ) SNP haplotypes (coding for a K residue atposition 232) appear to be in strong linkage disequilibrium (LD) withthe flanking microsatellite markers BULGE09 and BULGE11, as they are inessence associated with unique microsatellite haplotypes correspondingrespectively to the previously defined μH^(Q-D) and μH^(Q-NZ) haplotypes(FIG. 6C&D). In sharp contrast, the sh^(q) haplotype (coding for an Aresidue at position 232) is nearly evenly distributed across more thanten distinct microsatellite haplotypes (FIG. 6B).

These observations are in excellent agreement with the results of thecombined linkage and LD analysis (Fernier et. al., 2000). These studiesindeed predicted (i) that in the Dutch population the vast majority(estimates ranging from 81% to 92%) of “Q” allele (=K) would reside onthe μH^(Q-D) microsateflite haplotype, (ii) that in the New Zealandpopulation a large fraction (estimates ranging from 36% to 51%) of “Q”alleles would reside on haplotype μH^(Q-NZ) (we now see that theremainder correspond mainly to the μH^(Q-D) microsatellite haplotype)and (iii) that in both populations the “q” alleles (=A) would correspondto multiple marker haplotypes, corresponding to h^(q).

FIG. 7 illustrates the gain in LD signal that could be obtained in theDutch Holstein-Friesian grand-daughter design when adding the DGAT1polymorphisms to the previously available markers for proximal BTA14qand performing a joint linkage and LD multipoint analysis (Fernier et.al., 2000) using the sires “daughter yield deviations” (DYD (Van Radenand Wiggans, 1991) corresponding to half breeding values) for milk fatpercentage as phenotype. It can be seen that the lod score attributableto LD essentially doubles (from 3.7 to 7.8), and maximizes exactly atthe position of the DGAT1 gene. This result strongly supports the causalinvolvement of the DGAT1 gene in the QTL effect. The corresponding MLestimates of the “Q” to “q” allele substitution effect (α/2) (as definedin Falconer and Mackay, 1996), residual standard deviation (σ),population frequency of the “Q” allele (f_(Q)), number of generations tocoalescence (g) and heterogeneity parameter (ρ) were respectively 0.11%(α/2), 0.06% (σ), 0.20 (f_(Q)), 5 (g) and 0.84 (ρ).

Using the same Dutch Holstein-Friesian population, the additive effectof the DGAT1 K232A polymorphism on milk yield and composition wasexamined. The sons DYDs for milk yield (kgs), protein yield (kgs), fatyield (kgs), protein percentage and fat percentage, were analysed usinga mixed model including (i) a regression on the number of K alleles inthe genotype (0, 1 or 2), and (ii) a random polygenic componentestimated using an individual animal model and accounting for all knownpedigree relationships. Table 2 below, reports the obtained results. Itcan be seen that the K232A mutation has an extremely significant effecton the five analysed dairy traits. The proportion of the trait varianceexplained by this polymorphism in this population ranges from 8%(protein yield) to 51% (fat percentage), corresponding to between 10%(protein yield) and 64% (fat percentage) of the genetic variance (=QTL+polygenic).

Note that the proportion of the variance explained by the full model(1-r² _(error)) is of the order of 70% for the yield traits and 80% forthe percentage traits, which is in agreement with the knownreliabilities of the corresponding DYDs (Van Raden and Wiggans, 1991).An interesting feature of this QTL effect is that the “q” to “Q”substitution increases fat yield, while decreasing milk and proteinyield, despite the strong overall positive correlation characterizingthe three yield traits.

TABLE 2 Effect of the DGAT1 K232A mutation on sire's daughter yielddeviations (DYDs) for milk yield and composition. Trait α/2 r² _(QTL)p-value_(QTL) r² _(polygenic) r² _(error) Milk yield  −158 Kgs 0.185.00E−35 0.49 0.32 (Kgs) Fat yield  5.23 Kgs 0.15 1.57E−29 0.55 0.30(Kgs) Protein −2.82 Kgs 0.08 1.70E−15 0.65 0.26 yield (Kgs) Fat % 0.17%0.51 4.33E−122 0.29 0.19 Protein % 0.04% 0.14 5.05E−28 0.66 0.20 (i)α/2: QTL allele substitution effect on DYD (half breeding value),corresponding in the mixed model to the regression coefficient on thenumber of K alleles in the DGAT1 K232A genotype, and to α/2, where α isdefined according to ref. Falconer and Mackay, 1996. (ii) r² _(QTL):proportion of the trait variance explained by the DGAT1 K232Apolymorphism. (iii) p-value_(QTL): statistical significance of the DGAT1K232A effect. (iv) r² _(polygenic): proportion of the trait varianceexplained by the random, polygenic effect in the mixed model. (v) r²_(error): proportion of the trait variance unexplained by the model.The two previous analyses examined the effect of the DGAT1 polymorphismon estimated breeding values. By definition, this phenotype will onlyaccount for the additive component of the DGAT1 effect, and justifiesthe use of a regression on the number of K alleles in the mixed model.To evaluate the dominance relationship between the DGAT1 alleles, theeffect of the K232A genotype on the lactation values (first yielddeviations (Van Raden and Wiggans, 1991)) of the cows composing the NewZealand daughter design were analysed. This was achieved by using amixed model including (i) a fixed effect corresponding to the K232Agenotype, and (ii) a random polygenic component accounting for all knownpedigree relationships (“animal model”). Very significant effects ofK232A genotype on all examined yield and composition traits were foundin this population as well (Table 3, below), accounting for between 1%(protein yield) and 31% (fat percentage) of the trait variance. Theobserved dominance deviations, d, corresponding to the differencebetween the genotypic value of the KA genotype and the midpoint betweenthe AA and KK genotypic values (Falconer and Mackey, 1996) are shown inTable 3 below. Genotypic values of the heterozygous genotype aresystematically in between alternate homozygotes. None of the d-valuesproved to be significantly different from zero, indicating an absence ofdominance. Average K to A QTL allele substitution effects, α (Falconerand Mackey, 1996), were computed from the estimates of a- and d-values,as well as the population frequencies of the K and A alleles (Table 3).The predicted substitution effects are in good agreement with thosecomputed from the grand-daughter design: the K allele increases fatyield, fat % and protein %, while decreasing milk and protein yield. Theabsolute values of a estimated from the grand-daughter and daughterdesign are in perfect agreement for fat and protein %, while for theyield traits estimates are larger in the grand-daughter design whencompared to the daughter design. The exact reasons for this are beingexplored. It could be due to the fact that the sire population in thegrand-daughter design is not representative of the cow population ingeneral, or to intrinsic differences between the Dutch and New-Zealandpopulations and/or environment.

TABLE 3 Effect of the DGAT1 K232A mutation on cows′ lactation values formilk yield and composition. Trait a d α r² _(QTL) p-val_(QTL) r²_(polygenic) r² _(error) Milk yield (Kgs)  −144 Kgs   −42 Kgs  −161 Kgs0.03 1.05E−8 0.54 0.43 Fat yield (Kgs)  7.82 Kgs −0.89 Kgs  7.46 Kgs0.09 1.77E−20 0.46 0.45 Protein yield (Kgs) −2.34 Kgs −0.76 Kgs −2.64Kgs 0.01 4.35E−2 0.37 0.42 Fat % 0.41% 0.03% 0.42% 0.31  2.5E−108 0.490.20 Protein % 0.08% 0.03% 0.08% 0.04 1.60E−20 0.72 0.24 (i) a: half thedifference between the genotypic values of the KK and AA genotypes(Falconer and Mackey, 1996). (ii) d: dominance deviation (Falconer andMackey, 1996): deviation of the KA genotypic value from the midpointbetween the AA and KK genotypic values; none of these proved to besignificantly different from zero. (iii) α: average K to A substitutioneffect, computed as “a + d(q − p)” (Falconer and Mackey, 1996), where qis the allelic frequency of K (= 0.7) and p of A (= 0.3) (iv) r² _(QTL):proportion of the trait variance explained by the DGAT1 K232Apolymorphism. (v) p-val_(QTL): statistical significance of the DGAT1K232A effect. (vi) r² _(polygenic): proportion of the trait varianceexplained by the random, polygenic effect in the mixed model. (vii) r²_(error): proportion of the trait variance unexplained by the model.

Pedigree material and phenotypes. The pedigree material used for theassociation studies comprised a “grand-daughter design” (Weller et. al.,1990) counting 1,818 Holstein-Friesian bulls sampled in the Netherlands,as well as a “daughter-design” (Weller et. al., 1990) counting 529Holstein-Friesian cows sampled in New Zealand. The phenotypes of thesires were “daughter yield deviations” (DYD: unregressed weightedaverages of the daughter's lactation performances adjusted for 1systematic environmental effects and breeding values of the daughter'sdams and expressed as deviations from the population mean (Van Raden andWiggans, 1991)) obtained directly from CR-Delta (Arnhem—TheNetherlands). The phenotypes of the cows were “lactation values” (firstlactation yield deviations (YD), i.e. weighted average lactationperformances expressed as deviations from the population mean, adjustedfor management group, permanent environmental effects and herd-sireinteraction effects (Van Raden and Wiggans, 1991)) obtained directlyfrom Livestock Improvement Corporation (Hamilton—New Zealand).

Combined linkage and linkage disequilibrium analysis and associationstudies. The maximum likelihood procedure for combined linkage andlinkage disequilibrium analysis is described in detail in Farnir, 2000.The association study in the grand-daughter design was performed usingthe following model:Y _(i) =μ+βx _(i) +a _(i) +e _(i)where y_(i) is the DYD of son i, μ is the overall population mean, β isa fixed regression coefficient estimating the A to K allele substitutioneffect, x_(i) is an indicator variable corresponding to the number of Kalleles in the K232A genotype, a_(i) is a random polygenic componentaccounting for all known pedigree relationships (“animal model” Lynchand Walsh, 1997) and e_(i) is a random residual. The association studyin the daughter design was performed using the model:y _(i) =μ+g _(i) +a _(i) +e _(i)where y₁ is the lactation value of cow i, g_(i) is a fixed effectcorresponding to the DGAT1 genotype (KK, KA, or AA), a_(i) is a randompolygenic component accounting for all known pedigree relationships(“animal model” Lynch and Walsh, 1997) and e_(i) is a random residual.In both instances, maximum likelihood solutions for β, g_(i), α_(i),e_(i), σ² _(a), σ² _(e) were obtained using the MTDFREML program(Boldman et al, 1997).3. G Notyp T Sting and Analysis II

This summarises the genotype testing and subsequent analysis ofHolstein-Friesian, Jersey and Ayrshire animals in a separate populationfrom those presented in genotype testing and analysis I, above.

Progeny Tested Sires

Each year Livestock Improvement Corporation (New Zealand) progeny testsome 200-300 bulls per year. This entails the bulls being geneticallyevaluated on the basis of 50-85 daughters per sire. The sires areevaluated for milk fat, milk protein, milk volume and 20 non-productiontraits. Semen has been retained from all progeny tested sires since theearly 1970s. DNA was extracted from the semen and genotyped for theK232A DGAT1 polymorphism using the 7900 Taqman system (see Methodssection below).

Statistical analysis was undertaken on this dataset using RestrictedMaximum Likelihood (REML) and the average information algorithm (Johnsonand Thompson, 1995). The linear model included the fixed effects ofDGAT1 (3 classes; 0, 1 and 2 copies of the Q allele i.e. the K residue)and a covariate corresponding to the proportion of overseas genetics.The random effect was animal with a relationship matrix based on allknown relationships. Daughter yield deviations (DYDs), weighted averagesof a sire's daughter's lactation performances expressed as deviationsfrom the population mean (van Raden and Wiggans 1991) were used as thephenotypic measurement. The phenotypes were weighted by a weightingfactor based on the variance of the DYD for a son being:

${{Var}\mspace{14mu}{DYD}} = {\lbrack \frac{1 + {( {n - 1} )\frac{1}{4}h^{2}}}{n} \rbrack\sigma_{p}^{2}}$where Var DYD is the variance of son's DYD; n is the number of daughterscontributing to the DYD; h² is the heritability, which was taken as 0.35for yield traits.

The dataset was analysed separately for the 3 major breeds;Holstein-Friesian, Jersey and Ayrshire.

Seventeen hundred and thirteen Holstein-Friesian sires were included inthe analysis. The effect of the DGAT1 polymorphism was extremelysignificant for the three milk production traits (Table 4). With eachadditional Q allele the level of milk fat production increases byapproximately 6 kg per lactation, milk protein production decreases byapproximately 2.5 kg per lactation and milk volume decreases byapproximately 125 litres per lactation.

TABLE 4 Effect of the DGAT1 polymorphism on milk production in theHolstein-Friesian bull population (kilograms per lactation). Fat ProteinMilk qq 0 0 0 Qq 6.86 −2.13 −128 QQ 11.83 −4.80 −266 st. error 0.87 0.6824The effects for the Jersey and Ayrshire breeds were less significantthan those of the Holstein-Friesian breed but were consistent indirection of effects.Daughters for Milk Components

Data collection was integrated with LIC's herd testing service using asample of 102 herds involved in LIC's Sire Proving Scheme (SPS) in 1995.In addition to milk volume from herd testing, the concentrations of fat,crude protein (total nitrogen), casein, whey and lactose weredetermined. The data was collected from over 3,000 cows born in 1996 andfirst calving in the 1998 spring season, these being predominantly thedaughters of approximately 220 SPS bulls. The milk characteristics weremeasured at three herd tests on each cow, with each herd having a herdtest in each of the Sept/Oct, Nov/Dec and Jan/Feb periods. The MilkoscanFT120, which employs Fourier transform infrared spectrophotometry withenhanced milk calibrations (Foss Electric Application Note Nos. 95, P/N492280 and 102, P/N 578377), was used to determine the milk componentconcentrations.

Nine hundred and twelve daughters were genotyped for the DGAT1polymorphism using the OLA system. Analysis was undertaken using SAS(Statistics, Version 5, 1985) fitting a general linear model. The modelincluded sire and maternal grandsire as fixed effects, DGAT1polymorphism (3 classes; 0, 1 and 2 copies of the Q allele), covariatesincluding 16ths of Holstein-Friesian, Jersey, Ayrshire and other,proportion of overseas genetics within the Holstein-Friesian, Jersey andAyrshire breeds. Yield deviations that were pre-adjusted for herd, stageof lactation among other fixed effects were used (Johnson et al 2000).

The DGAT1 polymorphism is statistically significant for Lactose, casein,beta-casein and whey yield and also for casein and beta-casein percentas outlined in Table 5.

TABLE 5 Effect of the DGAT1 polymorphism on milk components. Trait qq QqQQ p-value Lactose yield* 48 23 0 <0.0001 Casein yield* 11.0 5.8 0 0.01Casein % −0.13 −0.06 0 <0.0001 Whey yield* 6.86 2.31 0 <0.0001 β-caseinyield* 3.98 2.19 0 0.05 β-casein % −0.43 −0.23 0 0.0001 *Units = g/dayfor lactose, casein and whey yield and g/litre for β-casein yieldDaughters for Solid Fat Content

Six hundred and ninety-two daughters were phenotyped for solid fatcontent. Solid fat content of the milkfat is a characteristic which hasa major influence on the functionality of milkfat products, and inparticular has a significant effect on the hardness of butter (MacGibbon& McLennan, 1987). The solid fat content at 10° C. (SFC 10) was used forcomparison of the properties of the milkfat as it relates well to thesectility hardness measurement of butter, a major functional property.Thus the performance of milkfat products may be predicted from thecharacteristics of the milk produced. The solid fat content (SFC) of theextracted fat was determined by pulsed nuclear magnetic resonance (NMR)and expressed as percentage solid fat (MacGibbon & McLennan, 1987). Asthe milkfat was melted to remove any thermal history, prior torecrystallization under standard conditions, the SFC simply reflects thechemical composition of the milkfat.

The 692 daughters were a subset of the 912 daughters that werephenotyped and genotyped for the results presented in Table 2. The solidfat content measures were collected over 2 lactations. Breeding valueswere calculated using an animal model similar to that of Johnson et al2000.

The same statistical model was fitted for solid fat content as was forthe milk component analysis. The DGAT1 polymorphism has a statisticallysignificant effect (p-value <0.0001) on solid fat content, increasing itby approximately by 1% for each addition of the Q allele.

This effect was further confirmed in 50 daughters (predominantlyHolstein-Friesian) that were farmed at one location and measured for SFCon the same day. The estimated effect for of the DGAT1 polymorphism onSFC was to increase it by approximately 2% per addition of each Qallele. This finding was significant at the five percent thresholdlevel.

The genetic standard deviation for SFC is 2.25 (D Johnson personalcommunication) and thus the effect of DGAT1 is approximately 0.5 of agenetic standard deviation.

4. Relative Transcript Levels of the Splice Variant

Real time PCR experiments were conducted using reverse transcribed mRNAisolated from lactating bovine mammary gland(s) (see experimentalmethods). These experiments revealed that the alternatively splicedtranscript as shown on FIG. 2 b, was approximately 100 fold lessabundant than the full length transcript.

METHODS SECTION

In order to identify other polymorphisms within the bovine DGAT1 gene,DNA was isolated from sperm, PCR amplified and then using primersdesigned from the sequence shown in FIGS. 2 a and 2 b and/or the cDNAsequence (SEQ ID NO: 4) direct sequenced on an ABI 3100. The breedsexamined were:

Ayrshire, Angler, Belgian Blue, Blond D'Aquitaine, Brown Swiss,Charolais, Red Devon, Devon, Dexter, Friesian, Guernsey, BeltedGalloway, Gelbvieh, Hereford, Jersey, Limousin, Longhorn, Maine Anjou,MRI (Meuse-rhine-yssel), Murray Grey, Piedmontese, Romangola, Sahiwal,Santa Gertrudis, Scottish Highland, Shorthorn, South Devon, Sussex,Swedish Red, Simmental, Wagyu, Welsh Black, Angus, and Zebu.

All the polymorphisms discovered are listed in Table 1, above.

The majority of the primers are also listed in FIG. 2 b or contained inthe cDNA sequence (SEQ ID NO: 4).

Experimental Method for the OLA Analysis of Four SNP's in DGAT1

PCR Amplification of the Regions Containing the Polymorphisms

Protocol for the PCR amplification of exon VIII, intron XII and 3′ UTR,the regions containing the four polymorphisms that were initiallydescribed in the DGAT1 gene.

Component For 1 sample Final concentration HotStar Qiagen Buffer (10 x) 1.5 μl  0.7 μM Primer 17F at 100 μM 0.07 μl  0.7 μM 18R at 10 μM 0.07μl  0.5 μM Primer 6F at 100 μM 0.05 μl  0.5 μM AW 446985dn1 at 100 μM0.05 μl  0.5 μM Primer InsUp1 0.05 μl  0.5 μM 14R2 0.05 μl 10% DMSO   1μl  300 μM dNTP 10 mM  0.3 μl  0.1 U/μl HotStarQiagen Taq  0.2 μl (CatNr203205: 5 U/μl) H2O 1.66 μl DNA (5 ng/μl)   5 μl Total   10 μl

Primer sequences are given in the following table as well as the genomicregion targeted by them.

SNP targeted Primer name Primer sequence SEQ ID No: 5′ base positionExon VIII SNP (DG 1) 17F CCTGAGCTTGCCTCTCCCACAGT 48 6579 18RCCAGGAGTCGCCGCAGCAGGAAG 49 7058 Exon XII SNPs 6FCCGGCCATCCAGAACTCCATGAAG 50 7280 (DG 2 and DG3) AW446985 dn1TAGAACTCGCGGTCTCCAAAC 51 7605 InsUp1 TGGCTGTCACTCATCATCGGGCA 52 82223′UTR SNP (DG4) 14R2 TTGCACAGCACTTTATTGACACA 53 8566

PCR amplification was performed on MJ PTC100 or PTCT200 cyclers usingthe following steps:

Step Temperature Time Comment 1° Activation 94° C. 12 minutes One timesof the enzyme 2° Denaturation 92° C.  1 minute Repeat step 3°Hybridisation 60° C.  1 minute 30 seconds 2 to 4, 35 4° Elongation 72°C.  1 minute 30 seconds 5° Inactivation 99° C. 45 minutes of the enzymeOligonucleotide Ligation Assay (OLA)

The oligonucleotides used in the OLA multiplex reaction are given in thetable below.

The detection of each mutation relies on the use of twofluorescent-labelled oligonucleotide (SNPx_FAM and SNPx_HEX) and onecommon 3′ and 5′ phosphorylated, non-labelled oligonucleotide(SNPx_(—)2P)

Size of the 5′ base Number of spacer ligation Locus Oligo Sequence SEQID NO: position phosphoramidites product^(a) DG1 SNP1_FAM AGC TTT GGCAGG TAA GGC 54 6813 SNP1_HEX AGC TTT GGC AGG TAA GAA 55 6813 32 SNP1_2PGGC CAA CGG GGG AG 56 6831 0 DG2 SNP2_FAM GCT GGC GGT GAG TGA 57 7424SNP2_HEX GCT GGC GGT GAG TGG 58 7424 39 SNP2_2P CCT GCT GGG TGG GGA 597439 3 DG3 SNP3_FAM GCT GGG TGG GGA CGC 60 7442 SNP3_HEX GCT GGG TGG GGACGT 61 7442 29 SNP3_P GTG GGG GCG GGT GG 62 7457 0 DG4 SNP4_FAM TGC CCCAAC CTG GGT 63 8388 SNP4_HEX TGC CCC AAC CTG GGC 64 8388 36 SNP4_2P GCAGCA GGA GGA GGC 65 8403 2 ^(a)The size of the ligation products is thesum of the number of nucleotides of the two ligated oligonucleotidesplus 3 bases equivalents per spacer phosphorazmdites molecule, presentat the 5′ end of the common oligonucleotide.

For each SNP a mixture of the three oligonucleotides was prepared first,following the dilution guidelines in the table below.

Oligonucleotide Final SNP mixture to mix Quantity concentration DG1(oligo. mixture) SNP1_FAM 10 μM 10 μl 1 μM SNP1_HEX 10 μM 20 μl 2 μMSNP1_2P 10 μM 20 μl 2 μM H₂O 50 μl DG2 (oligo. mixture) SNP2_FAM 10 μM10 μl 1 μM SNP2_HEX 10 μM 20 μl 2 μM SNP2_2P 10 μM 20 μl 2 μM H₂O 50 μlDG3 (oligo. mixture) SNP3_FAM 10 μM 10 μl 1 μM SNP3_HEX 10 μM 20 μl 2 μMSNP3_2P 10 μM 20 μl 2 μM H₂O 50 μl DG4 (oligo. mixture) SNP4_FAM 10 μM10 μl 1 μM SNP4_HEX 10 μM 30 μl 3 μM SNP4_2P 10 μM 20 μl 2 μM H₂O 40 μl

The ligation reaction for one sample was performed as follow:

Quantity Component persample DG1 oligonucleotide mixture^(a) (35, 70 and70 nM)  0.7 μl DG2 oligonucleotide mixture^(a) (12.5, 25 and 25 nM) 0.25μl DG3 oligonucleotide mixture^(a) (12.5, 25 and 25 nM) 0.25 μl DG4oligonucleotide mixture^(a) (12.5, 37.5 and 25 nM) 0.25 μl DMSO   2 μlIncubation buffer of the Tsc DNA ligase (Roche, Cat Nr   2 μl 1 939 807or 1 939 815) Tsc DNA ligase   1 μl H₂O 8.55 μl Multiplex PCR (seeabove)   5 μl Total   20 μl ^(a)The final concentration of theoligonucleotides in the ligation reaction is given between parenthesis(SNPx_FAM, SNPx_HEX and SNPx_2P respectively)

The sample was submitted to the following temperature cycling program ina MJ PTC100 or PTC 200 PCR machine.

Step Temperature Time Comment 1° Initial 98° C.  2 minutes One timesdenaturation step 2° Denaturation 94° C. 30 seconds Repeat step 2 to 3,30 3° Hybridisation 45° C.  3 minutes times and ligation 5° Inactivation99° C. 45 minutes of the enzyme

Following the LCR, 20 μl of H₂O was added to the ligation reaction. To0.5 μl of the diluted ligation reaction, either 2 μl of loading bufferwas added, or 2 μl loading buffer containing TAMRA350 internal line sizestandard.

The loading buffer was composed as follows: 1 part of blue dextran (50mg/ml)/EDTA (25 mM) and 6 parts of formamide

The TAMRA350 containing loading buffer was composed as follows: 3 partsTAMRA350 (Applied Biosystems 401736; 8 nM), 10 parts of Blue dextran (50mg/ml)/EDTA (25 mM) and 60 parts of formamide.

TAMRA containing samples was placed alternately with TAMRA free sampleswhen loaded onto the sequencing gel, in order to ease the identificationof the lanes on the gel image.

The samples may require further dilution in order to avoid a too intensefluorescent signal on the sequencer. It is also very likely that fromone primer batch to another, oligonucleotides concentrations will needadjustment.

The samples were denatured for 5 minutes at 95° C. before loading. Thesamples were then loaded onto a 6% denaturing acrylamide gel onsequencer ABI 373 or a 4% gel on sequencer ABI 377.

In addition to the OLA assays referred to above, genotyping of the DGAT1polymorphism was carried out by utilizing two different techniques fordetection of PCR products.

Gel-based Genotyping Assay Primer sequences 5′ to 3′, genomic sequenceposition within SEQ ID NO:1 brackets: (SEQ ID NO:66) DGAT1 21:GTAGCTTTGGCAGGTAAGAA (6811) (SEQ ID NO:67) DGAT1 22:GGGGCGAAGAGGAAGTAGTA (6984) (SEQ ID NO:68) DGAT1 23:TGGCCCTGATGGTCTACACC (6613) (SEQ ID NO:69) DGAT1 24B:GGGCAGCTCCCCCGTTGGCCGC (6850)

The final reaction conditions were 1×Gold PCR buffer, 2.5mM MgCl₂(Applied Biosystems), 200 μM each dNTP (Roche), 600 nM DGAT1 21 andDGAT1 22, 400 nM DGAT1 23 and DGAT1 24B (Invitrogen), 10%dimethylsulphoxide (Sigma), 3 μl DNA template and 2.5 units AmpliTaqGold DNA polymerase (Applied Biosystems) in a total volume of 50 μl.

Cycling conditions were a 94° C. initial denaturation for 5 minutes,then 35 cycles of denaturation at 94° C. for 30 seconds, annealing at56° C. for 30 seconds, extension at 72° C. for 20 seconds followed byone extension cycle of 72° C. for 2 minutes.

Primer positions around polymorphism (in bold) on genomic sequence from6587 to 6986.

                 DGAT123    TGGC CCTGATGGTC TACACC TGCCTCTCCC ACAGTGGGCTCCGTGCTGGC CCTGATGGTC {right arrow over (TACA)}CCATCC TCTTCCTCAAGCTGTTCTCC TACCGGGACG TCAACCTCTG GTGCCGAGAG CGCAGGGCTG GGGCCAAGGCCAAGGCTGGT GAGGGCTGCC TCGGGCTGGG GCCACTGGGC TGCCACTTGC CTCGGGACCGGCAGGGGCTC GGCTCACCCC                  DGAT1 21 GTAGCT TTGGCAGGTA AGAACGACCCGCCC CCTGCCGCTT GCTCGTAGCT TTGGCAGGTA {right arrow over(AGAA)}GGCCAA                                  ←         CGCCGGTTCGGGGGAGCT GCCCAGCGCA CCGTGAGCTA CCCCGACAAC CTGACCTACC GCCCCCTCGA CGGGDGAT1 24B GCGGTGAGGA TCCTGCCGGG GGCTGGGGGG ACTGCCCGGC GGCCTGGCCTGCTAGCCCCG CCCTCCCTTC CAGATCTCTA CTACTTCCTC TTCGCCCCCA (SEQ ID NO:70                    ←      AT GATGAAGGAG AAGCGGGG DGAT1 22The Q allele has polymorphic sequence AA and is detected by the DGAT121+22 primers, producing a band of 174 bp. The q allele has polymorphicsequence GC and is detected by the DGAT123+24 primers, producing a bandof 238 bp.

The primers DGAT123 and DGAT122 also successfully PCR the DGAT1 geneproducing a product of 372 bp in all reactions. Therefore, a QQhomozygote would have bands at 372 bp and 174 bp, a qq homozygote wouldhave bands at 372 bp and 238 bp and a Qq heterozygote would have all 3bands at 372 bp, 238 bp and 174 bp.

18 μl of PCR product was separated on a 1.2% agarose TAE gel, stainedwith ethidium bromide and scored independently by two investigators onthe basis of the number and size of bands present.

TaqMan Allelic Discrimination Genotyping Assay Primer sequences 5′ to3′, genomic sequence position in brackets: (SEQ ID NO:71) DGAT1forAD:TTCTCCTACCGGGACGTCAA (6651) (SEQ ID NO:72) ReverseNZ:CCGCGGTAGGTCAGGTTGTC (6890) Probe sequences 5′ to 3′, genomic sequenceposition in brackets: (SEQ ID NO:73) ForAA (FAM): CGTTGGCCTTCTTA (6838)(SEQ ID NO:74) DGAT1ADGC (VIC): TTGGCCGCCTTACC (6836)Both probes use MGB (minor groove binder) as a non-fluorescent quencher.

The final reaction conditions are 1× Universal PCR Mastermix (AppliedBiosystems), 500 nM each primer (Invitrogen), 70 nM ForAA (FAM) probe,300 nM DGAT1ADGC (VIC) probe (Applied Biosystems) and 2 μl of a 1/20dilution of DNA template in a total volume of 10 μl.

Cycling conditions were 50° C. for 2 minutes, 95° C. initialdenaturation for 10 minutes, then 37 cycles of denaturation at 94° C.for 15 seconds, annealing and extension 60° C. for 1 minute.

Primer positions around polymorphism (in bold) on genomic sequence from6587 to 6986 of SEQ ID NO:1.

TGCCTCTCCC ACAGTGGGCT CCGTGCTGGC CCTGATGGTC TACACCATCC     DGAT1forADTTCTCC TACCGGGACG TCAA TCTTCCTCAA GCTGTTCTCC TACCGGGACG TCAA{right arrowover (CCT)}CTG GTGCCGAGAG CGCAGGGCTG GGGCCAAGGC CAAGGCTGGT GAGGGCTGCCTCGGGCTGGG GCCACTGGGC TGCCACTTGC CTCGGGACCG GCAGGGGCTC GGCTCACCCC                       F rAA(FAM)A T TCTTCCGGTTGC CGACCCGCCC CCTGCCGCTTGCTCGTAGCT TTGGCAGGTA AGAAGGCCAA                          DGAT1ADGC(VIC)CCAT TCCGCCGGTT CGGGGGAGCT GCCCAGCGCA CCGTGAGCTA CCCCGACAACCTGACCTACC                          ←        CTGTTG GACTGGATGGGCGGTGAGGA TCCTGCCGGG GGCTGGGGGG ACTGCCCGGC GGCCTGGCCT CGCC ReverseNZGCTAGCCCCG CCCTCCCTTC CAGATCTCTA CTACTTCCTC TTCGCCCCCA (SEQ ID NO:75)

A 240 bp product is produced in this reaction. When the Q allele (AA) ispresent the FAM-labelled probe binds and fluoresces at 518 nm. When theq allele (GC) is present the VIC-labelled probe binds and fluoresces at554 nm. After cycling is complete, the plate is scanned on the ABI7900Sequence Detection System, the fluorescence from each well detected, anda scattergraph is drawn. The scattergraph separates out into 3 clumpswith Q homozygotes in the upper left hand corner, q homozygotes in thelower right hand corner and Qq heterozygotes in between. Each clump iscircled and the software automatically determines the genotype for eachsample. On each plate there are controls with 8 wells each of known Qhomozygotes, q homozygotes, Qq heterzygotes and no template controls.

Splice Variant Gene Expression

To determine the relative gene expression of the splice variants createdby insertion/deletion of 66 bp around the polymorphic site by alternateexon usage, RNA was extracted from mammary tissue and reversetranscribed using oligodT primer using a first strand cDNA synthesis kit(Invitrogen). Real time PCR to determine relative quantities of eachvariant was then carried out.

Primer sequences 5′ to 3′, genornic sequence position in brackets: (SEQID NO:76) DGAT1forRT66: TCTCCTACCGGGACGTCAAC (6652) (SEQ ID NO:77)DGAT1revRT66: GAGATCGCGGTAGGTCAGGTT (6964) (SEQ ID NO:78)DGAT1forRTless66: GCTGCTTTGGCAGATCTCTACTACTT (6711) (SEQ ID NO:79)DGAT1revRTless66: AAGCGCTTTCGGATGCG (7038) Probe sequences 5′ to 3′,genomic sequence position in brackets: (SEQ ID NO:80) DGAT1with66 (FAM):CCGTGAGCTACCC (6857) (SEQ ID NO:81) DGAT1less66 (VIC): CTTCGCCCCCACCCT(6976)Both probes use MGB (minor groove binder) as a non-fluorescent quencher.

Final reaction conditions were 1×Universal PCR Mastermix (AppliedBiosystems), 60 nM each primer (Invitrogen), 60 nM each probe (AppliedBiosystems) and 1 μl of template cDNA in a total volume of 10 μl.

Cycling conditions were 50° C. for 2 minutes, 95° C. initialdenaturation for 10 minutes, then 37 cycles of denaturation at 94° C.for 15 seconds, annealing and extension 60° C. for 1 minute.

Primer positions around 66 bp insertion (in italics) on cDNA sequence.The start of the cDNA sequence is equivalent to position 6479 on thegenomic sequence, with the last base of the cDNA equivalent to position7428 of the genomic sequence.

CCGTGGCCTT TCTCCTCGAG TCTATCACTC CAGTGGGCTC CGTGCTGGCC                         DGAT1forRT66 TCTCCT ACCGGGACGT CTGATGGTCTACACCATCCT CTTCCTCAAG CTGTTCTCCT ACCGGGACGTCAAC→                           DGAT1forRTless66 GCTGCTT CAACCTCTGGTGCCGAGAGC GCAGGGCTGG GGCCAAGGCC AAGGCTGCTTTGGCAG                    DGAT1with66(FAM) C CGTGAGCTAC TGGCAGGTAAGAAGGCCAAC GGGGGAGCTG CCCAGCGCAC CGTGAGCTACCC                           ATCTCTAC TACTT→ CCCGACAACC TGACCTACCGCGATCTCTAC TACTTCCTCT TCGCCCCCAC   ←TTGG ACTGGATGGC GCTAGAGDGAT1revRT66CT TCGCCCCCAC CCTGTGCTAC GAGCTCAACT TCCCCCGCTC CCCCCGCATC CGAAAGCGCT CCTDGAT1less66 (VIC)       ←     GCGTAG GCTTTCGCGA TCCTGCTGCG GCGACTCCTGGAGATGCTGT TCCTCACCCA GCTCCAGGTG A DGAT1revRTless66 GGGCTGATCCAGCAGTGGAT GGTCCCGGCC ATCCAGAACT CCATGAAGCC CTTCAAGGAC ATGGACTACTCCCGCATCGT GGAGCGCCTC CTGAAGCTGG (SEQ ID NO:82)This reaction detects the presence of the insertion splice variant bycreating a 145 bp product which binds the FAM probe only. The deletionsplice variant is detected by a 92 bp product that binds the VIC probeonly.

The cDNA for each alternate splice variant was cloned into pGemT(Promega). A dilution series of the same, known amount, of each variantplasmid DNA was used to create a standard curve that established thelinearity of the PCR reaction over a range of DNA concentrations. Thethreshold cycle number of the sample variants was converted back to aDNA amount by linear regression and the amounts of each variant presentcompared.

The presence of an alternate spice variant raises the possibility of analternate function that is at this stage unknown.

It will be appreciated that it is not intended to limit the invention tothe above examples only, many variations, which may readily occur to aperson skilled in the art, being possible without departing from thescope thereof as defined in the accompanying claims.

INDUSTRIAL APPLICATION

The present invention is directed to a method of genotyping bovine forimproved milk production traits. In particular, such traits includeincreased milk volume and milk protein content and decreased milkfatcontent and solid fat content. It is anticipated that herds of bovineselected for such a trait will produce milk which will be more easilyprocessed and such milk and products made therefrom may provide healthbenefits to consumers, as well as producing an increased milk yield.

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1. A method of identifying a bovine, or sample derived from said bovine,with a nucleic acid composition indicative of increased milk volumeproduction in said bovine, the method comprising the steps of: (a)providing nucleic acids from said bovine, or a sample derived from saidbovine where said sample comprises nucleic acids from said bovine; (b)detecting, in said nucleic acids, the presence of nucleotides G and C atpositions 6829 and 6830 respectively in SEQ ID NO: 1; and (c)identifying said bovine or said sample with nucleotides G and C atpositions 6829 and 6830 respectively in SEQ ID NO: 1 as one thatpossesses a nucleic acid composition indicative of increased milk volumeproduction.
 2. The method of claim 1, wherein detection of the presenceof the nucleotides G and C is made via amplification of a nucleic acidsequence comprising the nucleotides.
 3. The method of claim 2, whereinprimers consisting of SEQ ID NOs: 20 and 21 are used in theamplification.
 4. The method of claim 1, wherein detection of thepresence of the nucleotides G and C is made using an oligonucleotideligation assay (OLA).
 5. The method of claim 4, wherein the OLA isperformed using at least one primer consisting of the sequence of SEQ IDNO: 54 or
 56. 6. The method of claim 1, in which detection of thenucleotides G and C is made via hybridization of a probe consisting of asequence complementary to at least 5 contiguous nucleotides of thesequence or complement of SEQ ID NO: 1 that comprises said G and C,wherein the probe is capable of hybridization to said sequence orcomplement of SEQ ID NO: 1 in 6x sodium citrate/sodium chloride (SSC) at45° C.
 7. A method of selecting a bovine with a nucleic acid compositionindicative of increased milk volume production, the method comprisingthe steps: (i) identifying a bovine, with a nucleic acid compositionindicative of altered milk volume production, by the method of claim 1,and (ii) selecting the bovine, identified in step (i).
 8. A method ofidentifying a bovine, or sample derived from said bovine, with a nucleicacid composition indicative of decreased milk volume production in saidbovine, the method comprising the steps of: (a) providing nucleic acidsfrom said bovine, or a sample derived from said bovine where said samplecomprises nucleic acids from said bovine; (b) detecting, in said nucleicacids, the presence of nucleotides A and A at positions 6829 and 6830respectively in SEQ ID NO: 1; and (c) identifying said bovine or saidsample with nucleotides A and A at positions 6829 and 6830 respectivelyin SEQ ID NO: 1 as one that possesses a nucleic acid compositionindicative of decreased milk volume production.
 9. The method of claim8, wherein detection of the presence of the nucleotides A and A is madevia amplification of a nucleic acid sequence comprising the nucleotides.10. The method of claim 9, wherein primers consisting of SEQ ID NOs: 20and 21 are used in the amplification.
 11. The method of claim 8, whereindetection of the presence of the nucleotides A and A is made using anoligonucleotide ligation assay (OLA).
 12. The method of claim 11,wherein the OLA is performed using at least one primer consisting of thesequence of SEQ ID NO: 55 or
 56. 13. The method of claim 8, in whichdetection of the nucleotides A and A is made via hybridization of aprobe consisting of a sequence complementary to at least 5 contiguousnucleotides of the sequence or complement of SEQ ID NO: 1 that comprisessaid A and A, wherein the probe is capable of hybridization to saidsequence or complement of SEQ ID NO: 1 in 6x sodium citrate/sodiumchloride (S SC) at 45° C.
 14. A method of selecting a bovine with anucleic acid composition indicative of decreased milk volume production,the method comprising identifying a bovine by the method of claim 8 andselecting the bovine that is identified.